本发明属于植物基因工程
技术领域：
：，具体涉及对乙烯不敏感棉花的构建及在抗黄萎病中应用。通过削弱乙烯信号的传导构建乙烯不敏感棉花材料，该材料可在棉花抗黄萎病育种中应用。本发明涉及一个atctr1基因，它是拟南芥乙烯信号路径中的负调控因子，缺失该基因的拟南芥突变体有组成型“三重反应”的表型。在陆地棉(gossypiumhirsutum)“yz1”中超量表达该基因可以降低棉花对乙烯的敏感性，增强棉花对黄萎病(verticilliumwilt)的抗性，降低发病棉株叶片的脱落率。本发明还涉及一个gheins基因，该基因是陆地棉乙烯信号路径中的正调控因子，利用vigs技术在陆地棉“yz1”中抑制gheins的表达能够增强棉花对黄萎病的抗性，降低发病棉株叶片的脱落率。本发明还涉及含有atctr1/gheins基因或其同源基因的载体，以及涉及利用atctr1/gheins基因或通过乙烯信号路径上的其他基因削弱乙烯信号的传导从而增强棉花对黄萎病抗性和抑制黄萎病发生导致棉花的落叶。
背景技术：
：：棉花是我国战略物资，用途广泛。但是我国棉花经常因棉花黄萎病而减产，其成为当前阻碍我国棉花生产的重大问题之一。棉花黄萎病是一种土传病害，其特点是病原菌从棉花根部侵入，之后在维管束中拓展，数天之后棉花叶片出现黄化萎蔫的症状，以及伴随有取决于病原菌的程度不同的脱落。相对于非落叶型黄萎病菌，落叶型黄萎病菌通常有较强的致病力和致落叶性。1983年，陆家云等首次证实了我国江苏省局部地区存在棉花落叶型黄萎病菌(陆家云等,江苏省棉花黄萎病菌(verticilliumdahliae)致病力的分化.南京农业大学学报,1983.6(1):p.36-43.)，随后我国黄河流域棉区、西北内陆棉区、长江流域棉区也陆续发现了落叶型黄萎病菌。近年来，棉田分离的黄萎病菌菌系中，落叶型菌系的比例逐渐提高(惠慧等,落叶型棉花黄萎病菌的致病力分化.植物保护学报,2013.40(5):p.445-449.)。目前新疆发生棉花黄萎病的棉田超过50％，且病情指数5.0以上的棉田占比高达28.1％；所采集分离的棉花黄萎病菌系中，37.3％为落叶型菌系，分离到棉花黄萎病菌系的区域中60％检测到落叶型菌系，在新疆棉区呈现普遍发生的状况(刘海洋等,新疆主要棉区棉花黄萎病发生概况.植物保护,2015.41(3):p.138-142.)。通常被落叶型黄萎病菌入侵的棉株在适宜条件下发病比较急促，伴随有快速的落叶，当后期温度不再适合黄萎病菌生长时，其恢复能力也较差，因而对棉花产量影响极大。找到落叶型菌系致落叶的因素可以为防治黄萎病提供参考。乙烯可以促进植物组织的脱落，张婷等发现黄萎病菌落叶型菌系v592乙烯生成量远高于非落叶型菌系v774，且落叶型菌系v592在棉花根系诱导下乙烯生成量进一步增加，而非落叶型菌系v774在棉花根系诱导下乙烯生成量变化较小(张婷等,不同致病力棉花黄萎病菌乙烯含量及相关基因表达量的测定.石河子大学学报(自科版),2016,34(5).)；wiese等发现落叶型菌系t9接种的棉株乙烯产生量明显高于非落叶型菌系ss4接种的棉株(wiese等,growthregulatorchangesincottonassociatedwithdefoliationcausedbyverticilliumalbo-atrum.plantphysiology,1970,45(3):304.)，暗示着落叶型菌系黄萎病菌导致的落叶和乙烯密切相关。当黄萎病菌中依赖camp蛋白激酶的催化亚基vdpkac1发生突变后，突变菌株生成乙烯的能力减弱，其对番茄的致病力也降低，表明黄萎病菌侵染植物时可能依赖于其自身乙烯的生成(tzima等,rolesofthecatalyticsubunitofcamp-dependentproteinkinaseainvirulenceanddevelopmentofthesoilborneplantpathogenverticilliumdahliae.fungalgenetics&biology,2010,47(5):406-415.)。申请人期望获得削弱乙烯信号传导的棉花材料探索乙烯在黄萎病菌导致落叶的作用以及在棉花抗黄萎病性中的功能，为生产上防治棉花黄萎病提供理论依据和技术支撑。乙烯的合成和信号传导在模式植物拟南芥中已经比较清楚。乙烯的合成始于甲硫氨酸(met)，met在s-腺苷甲硫氨酸合成酶催化下生成s-腺苷甲硫氨酸(adomet)，adomet在acc合成酶(acs)催化下生成1-氨基环丙烷-1-羧酸(acc)，acc在acc氧化酶(aco)催化下生成乙烯(yang等,ethylenebiosynthesisanditsregulationinhigherplants.annualreviewofplantphysiology,1984,35(1):155-189.)。乙烯信号的传导始于位于内质网膜上的受体结合乙烯，目前一共发现了5个乙烯受体，包括etr1及其同源基因ers1、ers2、etr2和ein4(chang等,arabidopsisethylene-responsegeneetr1:similarityofproducttotwo-componentregulators.science,1993.262(5133):p.539-44；hua等,ethyleneinsensitivityconferredbyarabidopsisersgene.science,1995.269(5231):p.1712-4；hua等,ein4anders2aremembersoftheputativeethylenereceptorgenefamilyinarabidopsis.plantcell,1998.10(8):p.1321-32.)。这些乙烯受体均需和下游的关键因子ctr1结合形成复合体负调控乙烯信号(gao等,localizationoftheraf-likekinasectr1totheendoplasmicreticulumofarabidopsisthroughparticipationinethylenereceptorsignalingcomplexes.journalofbiologicalchemistry,2003.278(36):p.34725-32.)。当受体没有结合乙烯时，其和ctr1形成的复合体可以磷酸化下游的ein2，ein2继而被泛素化降解，从而抑制乙烯信号的传导。当受体结合乙烯时，其构象改变，不再和ctr1结合成复合体，不再磷酸化下游的ein2，ein2蛋白得以积累。ein2蛋白的c端可以被切割下来，游离到细胞核中(ji等,fromendoplasmicreticulum(er)tonucleus:ein2bridgesthegapinethylenesignaling.molecularplant,2013.6(1):p.11-14.)，通过抑制ebf1/2介导的ein3/eil1泛素化降解而稳定ein3/eil1蛋白，ein3/eil1作为转录因子可以激活下游靶基因的转录(guo等,plantresponsestoethylenegasaremediatedbyscf(ebf1/ebf2)-dependentproteolysisofein3transcriptionfactor.cell,2003.115(6):p.667-77.)。ein5可以在mrna水平上抑制ebf1/2的表达，稳定ein3/eil1蛋白(olmedo等,ethylene-insensitive5encodesa5'-->3'exoribonucleaserequiredforregulationoftheein3-targetingf-boxproteinsebf1/2.procnatlacadsciusa,2006.103(36):p.13286-93.)。ein3/eil1靶基因erf1也是转录因子又能激活更下游基因的转录，产生乙烯反应。因此在乙烯信号路径中ctr1是信号路径传递的关键负调控因子，ein2是信号路径的正调控因子。增强ctr1的活性或者抑制ein2的活性均能消弱乙烯信号路径的传递，减弱乙烯调控植物生长发育的生理活性。因此申请人选择在棉花中超表达乙烯信号路径负调控因子ctr1以削弱乙烯反应。由于棉花中的ctr1基因功能没有得到验证，申请人通过异源表达拟南芥中的atctr1基因以期获得对乙烯敏感性降低的棉花材料。此外，通过分析，申请人还找到了棉花乙烯信号路径的正调控因子eins，通过抑制其表达水平能减弱乙烯的生理效应。在本发明提出之前，乙烯在棉花与黄萎病菌互作中的作用一直没有明确的结论。通过转基因手段将拟南芥乙烯信号路径上负调控基因atctr1导入到陆地棉“yz1”中，结果表明：超量表达该基因的植株较对照材料，对黄萎病抗性显著提高。同时，超量表达该基因的植株较对照材料，同时期落叶率显著下降。通过vigs的方法抑制陆地棉“yz1”的乙烯信号路径正调控基因gheins的表达水平，棉花幼苗的抗性也显著提高，落叶率显著下降。这表明申请人提出的通过削弱乙烯信号的传导提高棉花对黄萎病的抗性具有可行性，为未来培育抗黄萎病的棉花新品种提供了重要的理论基础和技术支撑。技术实现要素：本发明的目的是在于构建了超表达atctr1的载体并获得乙烯不敏感的棉花转基因材料，这种材料可在抗黄萎病中得到应用。该材料超表达了拟南芥的atctr1基因，其具有如seqidno:1所示的核苷酸序列，或至少50％同源性的序列，以及上述dna片段编码的蛋白或经过改造修饰的具有相同功能的蛋白。本发明的另一个目的是在于提供一种通过抑制乙烯信号路径上正调控基因gheins的表达，达到降低棉花植株对乙烯的敏感性，提高棉株对黄萎病抗性。可以使用包括本发明构建的超表达atctr1的乙烯不敏感棉花用于培育抗黄萎病的棉花品种。本发明的技术方案如下所述：从拟南芥中植物中提取总rna(提取方法根据zhu等，animprovedsimpleprotocolforisolationofhighqualityrnafromgossypiumspp.suitableforcdnalibraryconstruction,actaagronomicasinica.2005,31.1657-1659.)，利用反转录酶superscriptⅲ(购自invitrogen公司,美国)将其反转录合成cdna，反应条件为：65℃5min，50℃60min，70℃10min。根据atctr1已公布的cds序列信息设计引物atctr1-f(5’atggaaatgcccggtagaag3’)和atctr1-r(5’ttacaaatccgagcggttgg3’)扩增出atctr1基因的orf(2466bp)。pcr反应条件：94℃预变性3min；94℃30sec，59℃30sec，72℃2min30sec，32个循环；72℃延伸7min。将扩增获得的pcr产物连入pgem-t载体(购自promega公司，美国)，筛选阳性克隆并测序，获得所需的基因orf(编码阅读框，即cds)，该基因的核苷酸序列如seqidno：1所示，其编码的蛋白质的序列如seqidno:2所示。在上述atctr1-f和atctr1-r引物的两端分别加上saci和xbali酶切位点，分别将上述引物命名为atctr1oe-f(5’gagctcatggaaatgcccggtagaag3’)和atctr1oe-r(5’tctagattacaaatccgagcggttgg3’)，再以阳性克隆的质粒为模板进行pcr扩增，得到的pcr产物通过酶切连接反应构建到pcambia2300s载体上(一种超量表达转化载体，该载体为pcambia2300s，由澳大利亚cambia实验室centerfortheapplicationofmolecularbiologytointernationalagriculture惠赠的pcambia2300改造后得到。方法是：将camv35s启动子通过酶切连接反应整合到pcambia2300的多克隆位点上，即得到重组载体pcambia2300s，上述构建方法是本领域常用方法)，进一步从而获得超(过量)表达载体，命名为p35s-atctr1。该过量表达载体p35s-atctr1含有seqidno：1所示的核苷酸序列(图3)，以及植物表达载体pcambia2300s。采用农杆菌gv3101(roger等，aguidetoagrobacteriumbinarytivectors.trendsinplantscience.2000.5,1360-1385)介导的遗传转化棉花的方法和程序，并参照jin等建立的高效转化体系(identificationofanovelelitegenotypeforinvitrocultureandgenetictransformationofcotton.biologiaplantarum,2006,50:519-524；anefficientgraftingsystemfortransgenicplantrecoveryincotton(gossypiumhirsutuml.).plantcell,tissueandorganculture,85:181-185,2006；factorsaffectingstabletransformationandplantregenerationduringtransformingembryogeniccallusofuplandcotton(gossypiumhirsutuml.)viaagrobacteriumtumefaciens,plantcell,tissueandorganculture,81:229-237,2005)，将构建好的p35s-atctr1载体导入棉花材料“yz1”宿主细胞中。对转基因植株分单株提取叶片的rna，利用反转录酶superscriptⅲ(购自invitrogen公司,美国)将其反转录合成cdna，以引物atctr1-rtf(5’acaggagcaaccataacagcg3’)和atctr1-rtr(5’ttcggatcgtcggcacaagta3’)进行rt-pcr。pcr反应条件为：94℃预变性3min；94℃30sec，58℃30sec，72℃30sec，30个循环；72℃延伸7min。申请人在获得转基因高世代棉花品系后，对其进行了抗性鉴定。抗性鉴定的方法参考何昕等报道的棉花黄萎病抗性鉴定方法(he等,ghjaz2attenuatescottonresistancetobioticstressesviatheinhibitionofthetranscriptionalactivityofghbhlh171.molecularplantpathology,2017.)，利用黄萎病菌v991(一种强致病力落叶型黄萎病菌菌株，为常规获得)分别接种atctr1超量表达转基因棉花以及野生型(即非转基因)棉花品系“yz1”，结果发现atctr1超量表达转基因棉花对黄萎病菌的抗性较野生型(即非转基因)棉花品系“yz1”明显提高。在接菌后7天取样分析，发现atctr1超量表达的棉花株系乙烯信号路径相关基因的表达水平降低，与叶片脱落相关基因的表达明显低于野生型棉花品系“yz1”。这也说明接种黄萎病菌后脱落相关基因的表达是叶片脱落的重要原因，而削弱乙烯信号的传导可以降低这些基因的表达从而减少落叶。通过拟南芥中的ein2蛋白(genbank登陆号aed90580.1)在陆地棉蛋白数据库中(g.hirsutumnbiproteins)中进行比对，找到一个同源度很高的序列，其登陆号为gh_a09g1399，申请人将该基因命名为gheins。依据登陆号gh_a09g1399的序列设计引物用于vigs(病毒介导的基因沉默技术)载体的构建，以达到共同抑制基因表达的目的，在正向引物5’端加上bamhi酶切位点，反向引物5’端加上kpni酶切位点，具体引物的核苷酸序列如下：gheins-9vigss:5’cggggtacccaaagttgagcggggaga3’，gheins-9vigsa:5’cgcggatcctgcccaccaagattccag3’；利用vigs技术(gao等，proteomicandvirus-inducedgenesilencing(vigs)analysesrevealthatgossypol,brassinosteroidsandjasmonicacidcontributetotheresistanceofcottontoverticilliumdahliae.molecularcellularproteomics,2013,12:3690-3703.)抑制gheins表达后，发现棉花植株增强了对黄萎病的抗性，同时也降低了落叶率。从棉花叶片中提取总rna(根据zhu等，animprovedsimpleprotocolforisolationofhighqualityrnafromgossypiumspp.suitableforcdnalibraryconstruction,actaagronomicasinica.2005,31.1657-1659，报道的方法)，利用反转录酶superscriptⅲ(购自invitrogen公司,美国)通过反转录合成cdna。反应条件：65℃5min，50℃60min，70℃10min。之后根据以上引物利用pcr扩增得到相应的片段。pcr反应条件：94℃预变性3min；94℃30sec，56℃30sec，72℃35sec，32个循环；72℃延伸至7min。将扩增获得的pcr产物连入pgem-t载体(购自promega公司，美国)，筛选阳性克隆并测序，获得所需的片段。后经过bamhⅰ和knpⅰ双酶切和连接反应，将目标片段连接到trv载体(由荷兰，瓦赫宁根大学的bart.p.h.j.thomma教授惠赠)上，电击转化gv3101得到阳性菌株。本发明的优点：本发明从乙烯信号路径上的负调控基因ctr1和正调控基因ein2入手，探索乙烯在棉花与黄萎病菌互作中的作用。申请人发现削弱棉花对乙烯的敏感性，可以提高棉花对黄萎病的抗性，同时降低发病棉花的落叶率，这为控制棉花因黄萎病，导致减产提供了一种新的解决方案。本发明具体优点在于：1.乙烯在植物与黄萎病菌互作中的作用一直存在争议，本发明中较为明确的回答了乙烯在棉花与黄萎病菌互作中的作用。2.利用棉花中超表达乙烯信号路径上的负调控基因或抑制正调控基因的表达，削弱棉花对乙烯的敏感性，可以提高棉花对黄萎病的抗性。对乙烯不敏感的棉花材料将有助于增强棉花对黄萎病的抗性，从而降低发病率，提高棉花产量。3.削弱棉花对乙烯的敏感性，同时可以降低发病棉花的落叶率，可以减少棉花因叶片脱落带来光合产物的损失以及增强棉花后期的恢复能力，缓和黄萎病导致的棉花减产。4.通过在棉花中利用异源转化拟南芥负调控基因atctr1获得了棉花乙烯不敏感材料，证明了部分基因在不同物种间存在着功能的可转移性。附图说明序列表seqidno:1是atctr1基因的cds序列。序列表seqidno:2是atctr1基因的蛋白质序列。序列表seqidno:3是登陆号gh_a09g1399基因的cds序列。序列表seqidno:4是登陆号gh_a09g1399基因的蛋白质序列。图1：利用rt-pcr检测atctr1基因在不同转基因株系中的表达水平示意图。附图标记说明：atctr1基因为外源基因，所以在野生型(简称wt，即非转基因的棉花品系“yz1”)中不表达，在两个超表达株系oeatctr1-14和oeatctr1-42(以后简称c14和c42)中有着较高水平的表达，其中ghubq7基因(genbank登陆号登录号dq116441)为内参基因。图2：一种超量表达载体p35s-atctr1的构建示意图。附图标记说明：将atctr1基因全长orf经酶切连接反应构建到载体pcambia2300s上改造而成。图3：转atctr1基因材料对乙烯敏感度测试试验示意图。附图标记说明：在图3中，oeatctr1-14和oeatctr1-42两个株系在暗培养乙烯利处理下，下肧轴显著长于野生型(wt)，表明atctr1基因在棉花中依然保留了其功能，即削弱了乙烯信号的传导。图4：转atctr1基因材料接种黄萎病菌v991试验示意图。附图标记说明：图4中的a图是接种黄萎病菌v991后15天的材料。图4中的b图是接种黄萎病菌v991后第20天茎杆第5节位的菌量检测。图4中的c图是接种黄萎病菌v991后第20天时棉株第2-3节间茎段恢复培养的结果。图4中的d图是接种黄萎病菌v991后不同株系的病指(病情指数)统计。图4中的e图是接种黄萎病菌v991后不同株系的落叶率统计。结果表明，超量表达atctr1的两个株系如c14和c42较对照材料病指和落叶率均降低。图5：接种黄萎病菌v991第7天后与落叶相关基因的表达分析。附图标记说明：接种黄萎病菌v991后第7天时取棉株1，3叶位的离层做表达量分析，发现接菌后7天，c14和c42株系包括ghe1,3b1(1,3葡萄糖苷内切酶)、ghchi(内切几丁质酶)、ghgun1(β-d-木糖酶)和ghbxl(内切葡聚糖酶)在内的离层形成和落叶相关基因较野生型(wt)材料表达量降低。说明削弱棉花乙烯信号的传导可抑制叶片脱落相关基因的表达量。图6：利用vigs沉默技术gheins后接种黄萎病的试验示意图。附图标记说明：图6中的a图是接种黄萎病菌v991后第14天时的材料及沉默效果的检测。图6中的b图是接种黄萎病菌v991后第18天时棉花茎杆第5节位的菌量检测。图6中的c图是接菌后18天第2-3节间茎段恢复培养的结果。图6中的d图是接种黄萎病菌v991后不同株系的病指统计。图6中的e图是接种黄萎病菌v991后不同株系的落叶率统计。结果表明，抑制棉花中ein2s的表达后，棉花幼苗较对照材料的病指和落叶率均显著降低，其抗黄萎病性得到提高。具体实施方式实施例1：超量表达载体的构建和转化为了验证atctr1转入棉花后是否具有调控功能，申请人通过构建得到超量表达载体，进而转化棉花的下胚轴。根据本说明书《
发明内容》中得到atctr1的orf设计超表达引物，即在引物两端分别加上saci和xbali的酶切位点，该引物被分别命名为atctr1oe-f和atctr1oe-r，再以阳性克隆的质粒为模板进行pcr扩增，得到的pcr产物通过酶切连接反应构建到pcambia2300s载体上，取反应产物2μl利用热击的方法转入到大肠杆菌top10中，最后将复苏后的菌液涂到含有k+抗性的固体lb培养皿中，37℃过夜。挑取单菌落，摇菌培养后，进行菌液pcr扩增，以35s启动子碱基序列设计的引物作为上游引物(35s-f：5’-ctgacgtaagggatgacgc-3’)，以atctr1-r为下游引物。分别进行pcr反应，反应条件为：95℃5min，95℃30sec，59℃30sec，72℃2min30sec，30个循环，72℃10min，15℃forever。将扩增得到的pcr阳性的菌液送相关的商业公司进行测序。从序列正确的菌液中提取质粒，得到构建好的超量表达质粒载体p35s-atctr1(图谱见图2)，所述的过量表达载体p35s-atctr1含有seqidno：3所述的核苷酸序列(图2)。引物序列如下：atctr1oe-f：5’gagctcatggaaatgcccggtagaag3’，atctr1oe-r：5’tctagattacaaatccgagcggttgg3’。将构建的载体p35s-atctr1转化农杆菌菌株gv3101(roger等,aguidetoagrobacteriumbinarytivectors.trendsinplantscience.2000,5,1360-1385.)，以下农杆菌gv3101的来源相同，再通过农杆菌介导的转化方法转化棉花。转化棉花所采用的转化受体材料棉花品系“yz1”(河南省农科院经济作物研究所，又称豫早熟棉1号)参见：jin等的文献(identificationofanovelelitegenotypeforinvitrocultureandgenetictransformationofcotton.biologiaplantarum,2006,50:519-524.)。此材料是华中农业大学作物遗传改良国家重点实验室经过大量的筛选发现的一个具有很高胚胎发生能力的棉花材料，农杆菌介导的转化棉花的方法和程序参照jin等建立的高效转化体系(identificationofanovelelitegenotypeforinvitrocultureandgenetictransformationofcotton.biologiaplantarum,2006,50:519-524；anefficientgraftingsystemfortransgenicplantrecoveryincotton(gossypiumhirsutuml.).plantcell,tissueandorganculture,85:181-185,2006；factorsaffectingstabletransformationandplantregenerationduringtransformingembryogeniccallusofuplandcotton(gossypiumhirsutuml.)viaagrobacteriumtumefaciens,plantcell,tissueandorganculture,81:229-237,2005)，具体方法和流程如下所述：1、无菌苗的培养将将棉花品系yz1(参见：jin等的文献(identificationofanovelelitegenotypeforinvitrocultureandgenetictransformationofcotton.biologiaplantarum,2006,50:519-524)棉籽壳去掉，用0.1/100的升汞灭菌10min，无菌水冲洗三遍，接种于无菌苗培养基上(配方：ms大量元素25ml+葡萄糖15g+植物胶phytagel(购自sigma公司，美国)2.5g，补充蒸馏水至1l，调培养基的ph至6.1-6.2之间)，在28℃暗培养3-6天。2.农杆菌的活化和保存2.1材料和培养基、试剂的准备：农杆菌gv3101，制备含卡那霉素50mg/l的mgl液体培养基(配方：胰化蛋白胨5g，氯化钠5g，mgso4.7h2o0.1g,kh2po40.25g，甘露醇5g，甘氨酸1.0g，补充蒸馏水至1l，调培养基的ph至7.0)，无菌三角瓶，lb(固、液培养基为常规培养基)培养基(含卡那霉素50mg/l)，灭菌甘油，无菌枪头，无菌1.5ml离心管。2.2操作：2.2.1活化，悬浮：从超低温冰箱内取出保存的含有目标基因atctr1的gv3101菌株的甘油管在冰上融化，lb平皿上划线，26.5℃暗培养36-48h，待皿内长出清晰的单菌落，挑取单菌落在另外的lb平皿上划线，26.5℃暗培养36-48h，待皿内长出足够的菌落结束培养，把培养基表面菌落刮入三角瓶内的mgl培养基中，27℃、200rpm摇2h，当od值在0.5-1.5之间即可用于侵染。2.2.2菌株的保存：从培养皿内挑取单菌落接于lb液体培养基中150rpm，26℃摇48h，按菌液和甘油体积比为1：1加入1.5ml离心管混匀，-70℃保存。3.浸染，共培养：3.1准备：将暗培养5天左右幼嫩健壮的棉花品系yz1幼苗、活化的农杆菌、无菌培养皿和无菌滤纸等取出。3.2操作：无菌条件下将棉花品系yz1幼苗下胚轴用锋利的刀片切成0.5-1cm长的切段，转入到经活化的农杆菌菌液中，搅匀，静置5-10min，倒掉菌液，滤纸吸干残余菌液，吹5min使表面稍为干燥，分散布于垫有滤纸的共培养培养基中(配方：ms无机盐+b5有机物+2,4-d0.1mg/l+kt0.1mg/l+葡萄糖30g/l+phytagel2.5g/l，ph7.0)，20℃暗培养38-42h。4.愈伤组织的诱导将侵染共培养后的下胚轴切段接种于诱导培养基上(配方如下：ms无机盐+b5有机物+2,4-d0.1mg+kt0.1mg+葡萄糖30g+phytagel2.5g，补充蒸馏水至1l，调培养基的ph至5.8)。5、非胚性愈伤组织的增殖非胚性愈伤组织的增殖培养基配制：ms无机盐(其中的硝酸钾加倍，硝酸铵减半)+b5培养基的有机物+2,4-d0.05mg+kt0.1mg+葡萄糖30g+phytagel2.5g，补充蒸馏水至1l，调培养基的ph至5.8。6、愈伤组织的分化愈伤组织经继代(一个月继代一次)几次后，有的愈伤组织转成米粒状颗粒，将其转入分化培养基上(配方：ms基本培养基+b5培养基的有机物+激动素(kt)0.15mg+吲哚丁酸(iba)0.5mg+葡萄糖30g+phytagel2.5g，补充蒸馏水至1l，调培养基的ph至5.8)，进一步分化成胚状体。7、胚性愈伤组织的继代胚性愈伤组织的继代培养基如下所示：ms无机盐(其中硝酸钾加倍，硝酸铵减半)+b5培养基的有机物+kt0.15mg+iba0.5mg+gln(谷胺酰胺)1.0mg+天冬酰胺(asn)0.5mg+葡萄糖30g+phytagel2.5g，补充蒸馏水至1l，调培养基的ph至5.8。8、成苗生根培养将分化出的小苗继代到1/2ms培养基中成苗生长培养基上(配方：1/2ms无机盐+b5培养基的有机物+葡萄糖15g+phytagel2.5g，补充蒸馏水至1l，调培养基的ph至5.8)。9、炼苗移栽将生根良好的小苗揭开三角瓶封口膜，炼苗2-3天，然后移栽到小土钵中，遮荫缓苗一周左右移栽大田。实施例4：转基因材料棉花黄萎病菌的接种鉴定1、温室育苗选取籽粒饱满的棉花品系yz1的种子和转基因棉花的种子，将棉籽壳剥掉，用0.1％的升汞灭菌10min，无菌水冲洗三遍，接种于无菌苗培养基上(配方：ms大量元素25ml+葡萄糖15g+植物胶phytagel2.5g，补充蒸馏水至1l)，在28℃暗培养24-36h后，将胚根插入到培养基中，转到光下培养，设定温度为28℃，光照时间16h。2、棉花黄萎病菌v991的活化与培养将棉花黄萎病菌v991置于pda平板上(配方：称取200g马铃薯，洗净去皮切碎，加水1000ml煮沸15min，纱布过滤，再加15g葡萄糖和7g琼脂，充分溶解后趁热纱布过滤，在121℃高压蒸汽下灭菌30min)于25℃暗培养箱活化一周，用接种针取菌块放入到液体pda上(配制方法：称取200g马铃薯，洗净去皮切碎，加水1000ml煮沸15min，纱布过滤，再加15g葡萄糖充分溶解后趁热纱布过滤，在121℃高压蒸汽下灭菌30min)，然后将培养物黄萎病菌v991的pda在25℃、180rpm暗培养3-4d。2000rpm离心10min，将孢子重悬浮并把浓度调至107孢子/ml。3、棉花黄萎病菌接种待幼苗长到一到二叶期(营养液为常规hoagland氏营养液中)，选取生长一致的幼苗，将根系浸泡在浓度为107孢子/ml黄萎病菌孢子液中24h，之后再换为hoagland氏营养液。病情调查：参考何昕毕业论文中的方法(何昕,棉花多逆境响应基因的挖掘和功能验证[d].华中农业大学图书馆,2016)接菌后一周左右按照棉花黄萎病苗期分级标准调查并记载病情级数、计算病情指数，其中病情级数按温室棉苗发病的5级制记。0级：外表无病状。1级：病株叶片25％以下显病状。2级：叶片25％-50％显病状。3级：叶片50％以上显病状，有少数叶片凋落。4级：叶片全枯或脱落，生产力很低。落叶率(％)＝(叶片脱落数÷调查总叶片数)×100％。病指＝[各级病株数分别乘以相应级数之和÷(调查总株数×最高级数)]×100。接菌结果表明超表达atctr1基因的株系较对照棉花品系yz1的病指和落叶率均降低(图4)。利用vigs(gao等，proteomicandvirus-inducedgenesilencing(vigs)analysesrevealthatgossypol,brassinosteroidsandjasmonicacidcontributetotheresistanceofcottontoverticilliumdahliae.molecularcellularproteomics,2013,12:3690-3703.)抑制棉花gheins表达后，相比于对照其病指和落叶率降低(图6)，说明削弱乙烯信号的传导可以增强棉花对黄萎病的抗性和抑制黄萎病导致的棉花落叶。序列表<110>华中农业大学<120>对乙烯不敏感棉花的构建及在抗黄萎病中应用<141>2018-03-17<160>4<170>siposequencelisting1.0<210>1<211>2466<212>dna<213>棉花(gossypiumhirsutum)<220><221>gene<222>(1)..(2466)<220><221>cds<222>(1)..(2466)<400>1atggaaatgcccggtagaagatctaattacactttgcttagtcaattt48metglumetproglyargargserasntyrthrleuleuserglnphe151015tctgacgatcaggtgtcagtttccgtcaccggagctcctccgcctcac96seraspaspglnvalservalservalthrglyalaproproprohis202530tatgattccttgtcgagcgaaaacaggagcaaccataacagcgggaac144tyraspserleusersergluasnargserasnhisasnserglyasn354045accgggaaagctaaggcggagagaggcggatttgattgggatcctagc192thrglylysalalysalagluargglyglypheasptrpaspproser505560ggtggtggtggtggtgatcataggttg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